Azophenols as ERG oncogene inhibitors

ABSTRACT

Selective azophenol inhibitors of a wild type or an altered ERG protein expression are described, where the inhibitors represent a compound of Formula (I) or Formula (II) 
                         
wherein X, X 1 , X 2 , X 3 , X 4  and X 5 , R 1  through R 4  and R 9  are as described.

BACKGROUND OF THE INVENTION

The ETS Related Gene (ERG) proto-oncogene was characterized more thantwenty-five years ago (Rao et al., 1987a; Rao et al., 1987b; Reddy etal., 1987) and belongs to a large family of ETS transcription factorsthat are both positive and negative regulators of gene expression(Watson et al., 2010). These transcription factors are downstreameffectors of the mitogenic signal transduction pathways involved in cellproliferation, cell differentiation, development, transformation,apoptosis, and immune regulation (Watson et al., 2010; Sreenath et al.,2011; Dobi et al., 2013).

Prostate cancer (CaP) is the most frequently diagnosed non-skinmalignancy and second leading cause of cancer related deaths among menin the western countries, with a projected 1.7 million newly diagnosedcases worldwide (International Agency for Research of Cancer, WHO, PressRelease No 209, Mar. 21, 2012). An estimated 2.9 million patients in theUnited States and 11 million world-wide are currently living withprostate cancer(http://globocan.iarc.fr/old/FactSheets/cancers/prostate-new.asp;http://www.cancer.org/cancer/prostatecancer/detailedguide/prostate-cancer-key-statistics).While early detected CaP due to PSA screening is managed effectively bysurgery or radiation, a significant subset of CaP patients (20% to 40%)experience disease recurrence after definitive treatment and willrequire hormone ablation therapy (Eur. Urol. 2007 May; 51(5):1175-84,Epub 2007). Despite an initial response to therapy, metastatic CaPtumors eventually become refractory to hormone ablation therapy. Forthis subset of patients—i.e., those having metastatic hormone refractorycancer, there is no effective cure.

The ERG gene is the most prevalent and validated genomic alteration inprostate cancer The ERG proto-oncogene is overexpressed in 60-70% ofprostate tumors in patients of Caucasian ancestry as a result ofrecurrent gene fusions involving TMPRSS2 and the ETS family of genes(Petrovics et al., 2005; Tomlins et al., 2005; reviewed in Kumar Sinhaet al., 2008; Rubin et al., 2012). Emerging studies on human prostatecancer specimens and various experimental models underscore thecausative oncogenic function of ERG in prostate cancer (Klezovitch etal., 2008; Tomlins et al., 2008; Sun et al., 2008; Wang et al., 2008).ETS factors reprogram the androgen receptor cistrome and prime prostatetumorigenesis in response to PTEN loss (Chen et al., 2013; Nguyen etal., 2015) Numerous reports have highlighted both diagnostic andprognostic features of the genomic activation of ERG revealing thatabout half the prostate tumors harbor the most common gene fusion thattakes place between the androgen receptor-regulated TMPRSS2 genepromoter and ERG protein coding sequence (reviewed in Kumar-Sinha etal., 2008; Rubin et al., 2012). Fusion between the TMPRSS2 gene promoterand ERG results in the overexpression of N-terminally truncated orfull-length forms of ERG (Klezovitch et al., 2008; Sun et al., 2008;Sreenath et al., 2011). Fusion events between ERG and other androgeninducible promoter sequences, such as SLC45A3 (Han et al., 2008) andNDRG1 have also been identified in prostate cancer (Pflueger et al.,2009; Rubin et al., 2012).

ERG expression in CaP is androgen receptor (AR) dependent. While ARsignaling inhibitors are employed as therapeutics for treating CaP,compounds that selectively inhibit ERG expression are highly desirable.Up to 4 million of patients living with prostate cancer worldwide areexpected to harbor ERG positive tumors (Farrell et al., 2013). Compoundssuch as ERGi-USU are examples of such selective inhibitors that inhibitthe ERG protein in ERG positive cancer cell lines with minimal effect onnormal primary endothelial cells that endogenously express ERG—i.e., ERGnegative tumor or normal cells (PCT US2015/020172). The azophenols alsoselectively inhibit ERG expression and thus provide for the treatment ofcancers or pathologic conditions associated with an ERG fusion event orERG overexpression, including, for example, prostate cancer, Ewing'ssarcoma, acute myeloid leukemia, megakaryoblastic leukemia, endothelialcancer and acute T-lymphoblastic leukemia.

A systematic screening of 456 known kinases in kinase ligand competitionassays (Fabian et al., 2005) indicated potential ligands for RIO2(Kd=200 nM). The RIO family of atypical serine/threonine kinases wasfirst characterized in 1997 based on the studies of a right open readingframe (RIO1) gene, expressed constitutively at a low level inSaccharomyces cerevisiae (Angermayr et al., 1997). Unexpectedly, RIOkinase 2 (RIOK2) protein levels were observed to decrease in ERGexpressing VCaP cells in response to the azophenols of the inventionwith minimal effect on RIOK2 transcript levels as assessed by wholetranscriptome analyses. In the present invention, RIOK2 was investigatedas a potential target of the ERG inhibitors described herein.

SUMMARY OF THE INVENTION

An aspect of the present invention relates to a method for treating adisease associated with overexpression of wild type ERG protein, analtered ERG protein, ERG gene transcription or ERG mRNA translation in asubject suffering therefrom, comprising administering to the subject atherapeutically effective amount of a compound of Formula (I) or Formula(II)

or a pharmaceutically acceptable salt thereof, wherein:X is NH, O or S;X₁, X₂, X₃, X₄ and X₅ are independently N or CR₉, where only one of X₁,X₂, X₃, X₄ and X₅ is N;R₁, R₂ and R₄ are independently selected from the group consisting of H,aryl, halogen, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₃-C₇ cycloalkyl and C₃-C₇heterocycloalkyl, wherein the aryl, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₃-C₇cycloalkyl and C₃-C₇ heterocycloalkyl are optionally substituted withone or more substituents selected from the group consisting of C₁-C₈alkyl, C₃-C₇ cycloalkyl, C₃-C₇ heterocycloalkyl, aryl, heteroaryl,halogen, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OCHF₂, —OC₁-C₈ alkyl,—O-aryl, —O-heteroaryl, —NR₅R₆, —NR₅C(O)R₆ and —C(O)NR₅R₆;R₃ is selected from the group consisting of H, —OH, —NR₅R₆, C₁-C₁₀alkyl, C₁-C₁₀ alkoxy, C₃-C₇ cycloalkyl and C₃-C₇ heterocycloalkyl,wherein the C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, C₃-C₇ cycloalkyl and C₃-C₇heterocycloalkyl are optionally substituted with one or moresubstituents selected from the group consisting of C₁-C₈ alkyl, C₃-C₇cycloalkyl, C₃-C₇ heterocycloalkyl, aryl, heteroaryl, halogen, hydroxyl,—CN, —COOH, —CF₃, —OCH₂F, —OCHF₂, —OC₁-C₈ alkyl, —O-aryl, —O-heteroaryl,—NR₅R₆, —NR₅C(O)R₆ and —C(O)NR₅R₆;R₅ and R₆ are independently selected from the group consisting of H,C₁-C₈ alkyl, aryl and C₃-C₇ cycloalkyl, or R₅ and R₆ taken together forma C₃-C₇ heterocycloalkyl wherein the C₁-C₈ alkyl and C₃-C₇heterocycloalkyl are optionally substituted with one or moresubstituents selected from the group consisting C₁-C₈ alkyl, C₃-C₇cycloalkyl, C₃-C₇ heterocycloalkyl, aryl, heteroaryl, halogen, hydroxyl,—CN, —COOH, —CF₃, —OCH₂F, —OCHF₂, —OC₁-C₈ alkyl, —O-aryl, —O-heteroaryl,—NR₇R₈, —NR₇C(O)R₈ and —C(O)NR₇R₈;R₇ and R₈ are independently selected from the group consisting of H andC₁-C₈ alkyl;each R₉ is independently H, halogen, —CN, —OH, COOH, —NR¹⁰R¹¹, C₁-C₁₀alkyl, C₃-C₇ cycloalkyl, C₁-C₁₀ alkoxy and C₃-C₇ heterocycloalkylwherein the C₁-C₁₀ alkyl, C₃-C₇ cycloalkyl, C₁-C₁₀ alkoxy and C₃-C₇heterocycloalkyl are optionally substituted with one or moresubstituents selected from the group consisting of C₁-C₈ alkyl, C₃-C₇cycloalkyl, C₃-C₇ heterocycloalkyl, aryl, heteroaryl, halogen, hydroxyl,—CN, —COOH, —CF₃, —OCH₂F, —OCHF₂, —OC₁-C₈ alkyl, —O-aryl, —O-heteroaryl,—NR₁₀C(O)R₁₁ and —C(O)NR₁₀R₁₁;R₁₀ and R₁₁ are independently selected from the group consisting of H,C₁-C₈ alkyl and C₃-C₇ cycloalkyl, or R₁₀ and R₁₁ taken together form aC₃-C₇ heterocycloalkyl wherein the C₁-C₈ alkyl and C₃-C₇heterocycloalkyl are optionally substituted with one or moresubstituents selected from the group consisting C₁-C₈ alkyl, C₃-C₇cycloalkyl, C₃-C₇ heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl,—CN, —COOH, —CF₃, —OCH₂F, —OCHF₂, —OC₁-C₈ alkyl, —O-aryl, —O-heteroaryl,—NR₁₂R₁₃, —NR₁₂C(O)R₁₃ and —C(O)NR₁₂R₁₃; andR₁₂ and R₁₃ are independently selected from the group consisting of Hand C₁-C₈ alkyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; and R₁, R₂, R₃ and R₄ are independently selected from the groupconsisting of H and C₁-C₁₀ alkyl, such as C₁-C₈ alkyl, such as C₁-C₆alkyl, such as methyl, ethyl, isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; and at least one of R₁, R₂, R₃ and R₄ is not H.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₃ is H; and at least one of R₁, R₂ and R₄ is not H, such asC₁-C₁₀ alkyl, such as C₁-C₈ alkyl, such as C₁-C₆ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₄ is H; and at least one of R₁, R₂ and R₃ is not H, such asC₁-C₁₀ alkyl, such as C₁-C₈ alkyl, such as C₁-C₆ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₁ is H; and at least one of R₂, R₃ and R₄ is not H, such asC₁-C₁₀ alkyl, such as C₁-C₈ alkyl, such as C₁-C₆ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₃ and R₄ are each H; and at least one of R₁ and R₂ is not H, suchas C₁-C₁₀ alkyl, such as C₁-C₈ alkyl, such as C₁-C₆ alkyl, such asmethyl, ethyl, isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₁, R₃ and R₄ are each H; and R₂ is not H, such as C₁-C₁₀ alkyl,such as C₁-C₈ alkyl, such as C₁-C₆ alkyl, such as methyl, ethyl,isopropyl, butyl, isobutyl or pentyl.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; R₁, R₃ and R₄ are each H; and R₂ is halogen. In a particularembodiment, R₂ is fluorine, bromine or chlorine.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; each of R₂ and R₄ is H; R₁ is methyl; and R₃ is OH.

In an aspect of the invention directed to Formula (I), X is S; each R₉is H; each of R₁, R₂ and R₄ is H; and R₃ is OH.

In an aspect of the invention directed to Formula (I), X is NH; each R₉is H; each of R₁, R₂ and R₄ is H; R₁ is methyl; and R₃ is OH.

In an aspect of the invention directed to Formula (I), X is NH; each R₉is H; each of R₁, R₃ and R₄ is H; and R₂ is methyl or isopropyl.

In an aspect of the invention directed to Formula (I), X is O; each R₉is H; each of R₁, R₃ and R₄ is H; and R₂ is methyl or isopropyl.

In an aspect of the invention directed to Formula (I), X is O; each R₉is H; each of R₁, R₂ and R₄ is H; and R₃ is OH.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; and R₃ isOH.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₃ and R₄ is H; and R₂ ishalogen. In a particular embodiment, R₂ is fluorine, bromine orchlorine.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₂ and R₄ is H; R₁ is methyl;and R₃ is OH.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl.

In an aspect of the invention directed to Formula (II), X₂ is N; X₁, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; and R₃ isOH.

In an aspect of the invention directed to Formula (II), X₂ is N; X₁, X₃,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl.

In an aspect of the invention directed to Formula (II), X₃ is N; X₁, X₂,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; and R₃ isOH.

In an aspect of the invention directed to Formula (II), X₃ is N; X₁, X₂,X₄ and X₅ are CR₉; each R₉ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₃ is halogen and the R₉ ateach of positions X₂, X₄ and X₅ is H; each of R₁, R₂ and R₄ is H; and R₃is OH. In a particular embodiment, the halogen at X₃ is F, Cl or Br,such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₃ is halogen and the R₉ ateach of positions X₂, X₄ and X₅ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl pentyl or hexyl. In a particularembodiment, the halogen at X₃ is F, Cl or Br, such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₂ is halogen and the R₉ ateach of positions X₃, X₄ and X₅ is H; each of R₁, R₂ and R₄ is H; and R₃is OH. In a particular embodiment, the halogen at X₂ is F, Cl or Br,such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₂ is halogen and the R₉ ateach of positions X₃, X₄ and X₅ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl. In a particularembodiment, the halogen at X₂ is F, Cl or Br, such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₄ is halogen and the R₉ ateach of positions X₂, X₃ and X₅ is H; each of R₁, R₂ and R₄ is H; and R₃is OH. In a particular embodiment, the halogen at X₄ is F, Cl or Br,such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₄ is halogen and the R₉ ateach of positions X₂, X₃ and X₅ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl. In a particularembodiment, the halogen at X₄ is F, Cl or Br, such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₅ is halogen and the R₉ ateach of positions X₂, X₃ and X₄ is H; each of R₁, R₂ and R₄ is H; and R₃is OH. In a particular embodiment, the halogen at X₅ is F, Cl or Br,such as Br.

In an aspect of the invention directed to Formula (II), X₁ is N; X₂, X₃,X₄ and X₅ are CR₉, where the R₉ at position X₅ is halogen and the R₉ ateach of positions X₂, X₃ and X₄ is H; each of R₁, R₂ and R₄ is H; R₃ isNR₅R₆; and R₅ and R₆ are each independently C₁-C₈ alkyl, such as methyl,ethyl, isopropyl, butyl, isobutyl, pentyl or hexyl. In a particularembodiment, the halogen at X₅ is F, Cl or Br, such as Br.

In an aspect of the invention, the compound of Formula (I) is a compoundof Formula (III)

or a pharmaceutically acceptable salt thereof,wherein R₁ and R₂ are as defined for Formula (I).

In an aspect of the invention, R₁ and R₂ of Formula (III) areindependently selected from the group consisting of H, C₁-C₆ alkyl, suchas alkyl, such as methyl, ethyl, isopropyl, butyl and isobutyl, where atleast one of R₁ and R₂ is not H.

In an aspect of the invention, R₁ of Formula (III) is H and R₂ is C₁-C₆alkyl, such as C₁-C₄ alkyl, such as methyl, ethyl, isopropyl, butyl orisobutyl.

In an aspect of the invention, R₁ of Formula (III) is H and R₂ is C₁-C₆alkyl.

In an aspect of the invention, the compound of Formula (I) is

or a pharmaceutically acceptable salt thereof.

In an aspect of the invention, the compound of Formula (I) is

or a pharmaceutically acceptable salt thereof.

In an aspect of the invention, the compound of Formula (II) is acompound of Formula (IV)

or a pharmaceutically acceptable salt thereof,wherein R₃ and R₉ are as defined for Formula (II).

In an aspect of the invention, the compound of Formula (IV) is

or a pharmaceutically acceptable salt thereof.

In an aspect of the invention, the compounds of Formulae (I), (II),(III), (IV) and (V) are effective in treating a disease selected fromthe group consisting of prostate cancer, Ewing's sarcoma, acute myeloidleukemia, acute T-lymphoblastic leukemia, endothelial cancer and coloncancer.

In an aspect of the invention, the disease is prostate cancer.

Another aspect of the invention is a compound of Formula (V)

or a pharmaceutically acceptable salt thereof, whereinR₁ is selected from the group consisting of H and optionally substitutedC₁-C₁₀ alkyl, such as C₁-C₉ alkyl, such as C₁-C₈ alkyl, such as C₁-C₇alkyl, such as C₁-C₆ alkyl, such as C₁-C₅ alkyl, such as methyl, ethyl,isopropyl, butyl and isobutyl; andR₂ is optionally substituted C₂-C₁₀ alkyl, such as C₂-C₈ alkyl, such asC₂-C₇ alkyl, such as C₂-C₆ alkyl, such as C₂-C₅ alkyl, such as C₂-C₄alkyl, such as isopropyl, butyl and isobutyl, with the proviso that whenR₁ is H, R₂ is not ethyl, isobutyl or —C(CH₃)₂—CH₂—C(CH₃)₃.In an aspect of the invention, R₁ is H and R₂ is optionally substitutedC₂-C₁₀ alkyl, with the proviso that R₂ is not ethyl, isobutyl or—C(CH₃)₂—CH₂—C(CH₃)₃.

In an aspect of the invention, the compound of Formula (V) is

or a pharmaceutically acceptable salt thereof.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula (V)

or a pharmaceutically acceptable salt thereof, wherein:R₁ and R₂ are as defined for Formula (V).

In an aspect of the invention, the disease is prostate cancer.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound of Formula (I) and an excipient.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound of Formula (II) and an excipient.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound of Formula (III) and an excipient.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound of Formula (IV) and an excipient.

Another aspect of the invention is a pharmaceutical compositioncomprising the compound of Formula (V) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof Formula (I) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof Formula (II) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof Formula (III) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof Formula (IV) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising a compoundof Formula (V) and an excipient.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising co-administering to the subject a therapeuticallyeffective amount of a compound of Formula (I) in combination with atherapeutically effective amount of a known ERG inhibitor.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising co-administering to the subject a therapeuticallyeffective amount of a compound of Formula (II) in combination with atherapeutically effective amount of a known ERG inhibitor.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising co-administering to the subject a therapeuticallyeffective amount of a compound of Formula (III) in combination with atherapeutically effective amount of a known ERG inhibitor.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising co-administering to the subject a therapeuticallyeffective amount of a compound of Formula (IV) in combination with atherapeutically effective amount of a known ERG inhibitor.

An aspect of the invention is a method for treating a disease associatedwith overexpression of wild type ERG protein, an altered ERG protein,ERG gene transcription or ERG mRNA translation in a subject sufferingtherefrom, comprising co-administering to the subject a therapeuticallyeffective amount of a compound of Formula (V) in combination with atherapeutically effective amount of a known ERG inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are merely representative of selected embodimentsof the present invention and are not intended to define or narrow thescope of the invention as otherwise described herein. The chemicalstructures of Compounds 1 to 7 referred to in the Figures below aredepicted in FIG. 1A.

FIG. 1A depicts the azophenol compounds 1-13 for which testing as ERGinhibitors is disclosed herein. FIG. 1B represents exemplarysubstitution patterns for the compounds of Formula (I) as describedherein. FIGS. 1C and 1D depict the chemical structures of exemplaryazophenol compounds of Formula (I) and Formula (II) as described herein.

FIG. 2 illustrates the inhibition of endogenous AR, PSA and ERG proteinsin ERG harboring prostate cancer cells (VCaP) by Compounds 1 and 2 atconcentrations of 0.25 μM and 1 μM.

FIG. 3 illustrates the inhibition of the growth of ERG harboringprostate cancer cells (VCaP) by the Compounds 1 and 2 at concentrationsof 0.25 μM and 1 μM.

FIG. 4 illustrates the lack of inhibition of endogenous AR and PSAproteins in ERG negative prostate cancer cells (LNCaP) by Compounds 1and 2 at concentrations of 0.25 μM and 1 μM.

FIG. 5 illustrates that Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM do not affect the growth of ERG negative prostate cancer cells(LNCaP).

FIG. 6 illustrates that Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM do not inhibit endogenous AR protein in ERG negative prostatecancer cells (LAPC-4).

FIG. 7 illustrates that Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM do not affect the growth of ERG negative prostate cancer cells(LAPC-4).

FIG. 8 illustrates the inhibition of endogenous ERG and RIOK2 proteinsin ERG harboring cancer cells (COLO320) by Compounds 1 and 2 atconcentrations of 0.25 μM and 1 μM.

FIG. 9 illustrates the inhibition of growth of ERG harboring coloncancer cells (COLO320) by Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM.

FIG. 10 illustrates the lack of inhibition of ERG and RIOK2 proteins inendogenous ERG expressing normal endothelial cells (HUVEC) by Compounds1 and 2 at concentrations of 0.25 μM and 1 μM.

FIG. 11 illustrates that Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM do not affect the growth of ERG harboring normal endothelialcells (HUVEC).

FIG. 12 illustrates that Compounds 1 and 2 at concentrations of 0.25 μMand 1 μM do not affect the growth of ERG negative prostate-derivedimmortalized cells (BPH-1 and RWPE-1).

FIG. 13 illustrates the inhibition of ERG protein in VCaP cells byCompound 1 at concentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μMand 1 μM and shows the IC₅₀ of Compound 1 on ERG protein in VCaP.

FIG. 14 illustrates the inhibition of RIOK2 protein in VCaP cells byCompound 1 at concentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μMand 1 μM and shows the IC₅₀ of Compound 1 on RIOK2 protein in VCaP.

FIG. 15 illustrates the inhibition of VCaP cell growth by Compound 1 atconcentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM.

FIG. 16 illustrates the inhibition of ERG protein in VCaP cells byCompound 2 at concentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μMand 1 μM and shows the IC₅₀ of Compound 2 on ERG protein in VCaP.

FIG. 17 illustrates the inhibition of RIOK2 protein in VCaP cells byCompound 2 at concentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μMand 1 μM and shows the IC₅₀ of Compound 2 on RIOK2 protein in VCaP.

FIG. 18 illustrates the inhibition of VCaP cell growth by Compound 2 atconcentrations of 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM.

FIG. 19 illustrates the inhibition of VCaP cell growth by Compound 5 atconcentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM and shows theIC₅₀ of Compound 5 on ERG protein in VCaP.

FIG. 20 illustrates the inhibition of VCaP cell growth by Compound 6 atconcentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM and shows theIC₅₀ of Compound 6 on ERG protein in VCaP.

FIG. 21 illustrates the inhibition of VCaP cell growth by Compound 7 atconcentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM and shows theIC₅₀ of Compound 7 on ERG protein in VCaP.

FIG. 22 illustrates the inhibition of VCaP cell growth by Compound 5 atconcentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM and shows theIC50 of Compound 5 on cell growth.

FIG. 23 illustrates the inhibition of VCaP cell growth by Compound 6 atconcentrations of 0.02 μM, 0.05 μM, 0.10 μM, 0.20 μM and 0.40 μM andshows the IC50 of Compound 6 on cell growth.

FIG. 24 illustrates the inhibition of VCaP cell growth by Compound 7 atconcentrations of 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM and 1 μM and shows theIC50 of Compound 7 on cell growth.

FIG. 25 illustrates the inhibition of VCaP cell growth by Compound 3 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 3 on ERG protein in VCaP.

FIG. 26 illustrates the inhibition of VCaP cell growth by Compound 4 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 4 on ERG protein in VCaP.

FIG. 27 illustrates the inhibition of VCaP cell growth by Compound 5 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 5 on ERG protein in VCaP.

FIG. 28 illustrates the inhibition of VCaP cell growth by Compound 3 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 3 on VCaP cell growth.

FIG. 29 illustrates the inhibition of VCaP cell growth by Compound 4 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 4 on VCaP cell growth.

FIG. 30 illustrates the inhibition of VCaP cell growth by Compound 5 atconcentrations of 0.3 μM, 0.6 μM, 1.0 μM, 3.0 μM and 10.0 μM and showsthe IC₅₀ of Compound 5 on VCaP cell growth.

DETAILED DESCRIPTION Definitions

A “pharmaceutically acceptable salt” is a pharmaceutically acceptable,organic or inorganic acid or base salt of a compound of the invention.Representative pharmaceutically acceptable salts include, e.g., alkalinemetal salts, alkaline earth salts, ammonium salts, water-soluble andwater-insoluble salts, such as the acetate, amsonate(4,4-diaminostilbene-2, 2-disulfonate), benzenesulfonate, benzonate,bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium,calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate,einbonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts. Apharmaceutically acceptable salt can have more than one charged atom inits structure. In this instance the pharmaceutically acceptable salt canhave multiple counterions. Thus, a pharmaceutically acceptable salt canhave one or more charged atoms and/or one or more counterions.

The terms “treat”, “treating” and “treatment” refer to the ameliorationor eradication of a disease or symptoms associated with a disease. Incertain embodiments, such terms refer to minimizing the spread orworsening of the disease resulting from the administration of one ormore prophylactic or therapeutic agents to a patient with such adisease.

The terms “prevent,” “preventing,” and “prevention” refer to theprevention of the onset, recurrence, or spread of the disease in apatient resulting from the administration of a prophylactic ortherapeutic agent.

The term “effective amount” refers to an amount of a compound of theinvention, or other active ingredient sufficient to provide atherapeutic or prophylactic benefit in the treatment or prevention of adisease or to delay or minimize symptoms associated with a disease.Further, a therapeutically effective amount with respect to a compoundof the invention means that amount of therapeutic agent alone, or incombination with other therapies, that provides a therapeutic benefit inthe treatment or prevention of a disease. Used in connection with acompound of the invention, the term can encompass an amount thatimproves overall therapy, reduces or avoids symptoms or causes ofdisease, or enhances the therapeutic efficacy of or synergies withanother therapeutic agent.

A “subject” includes an animal, such as a human, cow, horse, sheep,lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit orguinea pig. The animal can be a mammal such as a non-primate and aprimate (e.g., monkey and human). In one embodiment, a subject is ahuman, such as a human infant, child, adolescent or adult.

The term “substituted”, as used herein, refers to the replacement of atleast one hydrogen atom of a molecular arrangement with a substituent.In the case of an oxo substituent (“═O”), two hydrogen atoms arereplaced. When substituted, one or more of the groups below are“substituents.” Substituents include, but are not limited to, halogen,hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl,alkoxy, alkylthio, haloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, heterocyclyl, and heterocycloalkyl; —NRaRb, —NRaC(O)Rb,—NRaC(O)NRaNRb, —NRaC(═O)ORb, —NRaSO₂Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)₂Ra, —OS(═O)₂Ra and—S(═O)ORa. In addition, the above substituents may be furthersubstituted with one or more of the above substituents, such that thesubstituent comprises a substituted alkyl, substituted aryl, substitutedarylalkyl, substituted heterocycle, or substituted heterocycloalkyl. Raand Rb in this context may be the same or different and, independently,hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heterocyclyl, substitutedheterocyclyl, heterocycloalkyl or substituted heterocycloalkyl.

The term “unsubstituted”, as used herein, refers to any compound thatdoes not contain extra substituents attached to the compound. Anunsubstituted compound refers to the chemical makeup of the compoundwithout extra substituents, e.g., the compound does not containprotecting group(s). For example, unsubstituted proline is a prolineamino acid even though the amino group of proline may be considereddisubstituted with alkyl groups.

The term “alkyl”, as used herein, refers to any straight chain orbranched, non-cyclic or cyclic, unsaturated or saturated aliphatichydrocarbon containing from 1 to 10 carbon atoms, while the term “loweralkyl” has the same meaning as alkyl but contains from 1 to 6 carbonatoms. The term “higher alkyl” has the same meaning as alkyl butcontains from 7 to 10 carbon atoms. Representative saturated straightchain alkyls include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and the like;while saturated branched alkyls include, but are not limited to,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Cyclic alkyls may be obtained by joining two alkyl groups bound to thesame atom or by joining two alkyl groups each bound to adjoining atoms.Representative saturated cyclic alkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include, but are not limited to, cyclopentenyland cyclohexenyl, and the like. Cyclic alkyls are also referred toherein as a “homocycles” or “homocyclic rings.” Unsaturated alkylscontain at least one double or triple bond between adjacent carbon atoms(referred to as an “alkenyl” or “alkynyl”, respectively). Representativestraight chain and branched alkenyls include, but are not limited to,ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl,2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include, but are not limited to, acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1-butynyl, and the like.

The term “aryl”, as used herein, refers to any aromatic carbocyclicmoiety such as, but not limited to, phenyl or naphthyl.

The term “arylalkyl”, or “aralkyl” as used herein, refers to any alkylhaving at least one alkyl hydrogen atom replaced with an aryl moiety,such as, but not limited to, benzyl, —(CH₂)₂-phenyl, —(CH₂)₃-phenyl,—CH(phenyl)₂, and the like.

The term “halogen”, as used herein, refers to any fluoro, chloro, bromo,or iodo moiety.

The term “haloalkyl”, as used herein, refers to any alkyl having atleast one hydrogen atom replaced with halogen, such as trifluoromethyl,and the like.

The term “heteroaryl”, as used herein, refers to any aromaticheterocycle ring of 5 to 10 members and having at least one heteroatomselected from nitrogen, oxygen and sulfur, and containing at least 1carbon atom, including, but not limited to, both mono and bicyclic ringsystems. Representative heteroaryls include, but are not limited to,furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, or quinazolinyl.

The term “heteroarylalkyl”, as used herein, refers to any alkyl havingat least one alkyl hydrogen atom replaced with a heteroaryl moiety, suchas —CHpyridinyl, —CH₂pyrimidinyl, and the like.

The term “heterocycle” or “heterocyclic ring”, as used herein, refers toany 3- to 7-membered monocyclic, or 7- to 10-membered bicyclic,heterocyclic ring which is either saturated, unsaturated, or aromatic,and which contains from 1 to 4 heteroatoms independently selected fromnitrogen, oxygen and sulfur, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized, including bicyclic rings in which any of theabove heterocycles are fused to a benzene ring. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles may includeheteroaryls exemplified by those defined above. Thus, in addition to theheteroaryls listed above, heterocycles may also include, but are notlimited to, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heterocycloalkyl”, as used herein, refers to any alkyl havingat least one alkyl hydrogen atom replaced with a heterocycle, such as—CH₂-morpholinyl, and the like.

The term “homocycle” or “cycloalkyl”, as used herein, refers to anysaturated or unsaturated (but not aromatic) carbocyclic ring containingfrom 3-7 carbon atoms, such as, but not limited to, cyclopropane,cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, andthe like.

The term “alkylamino”, as used herein, refers to at least one alkylmoiety attached through a nitrogen bridge (e.g., —N-alkyl or—N-(alkyl)-N—) including, but not limited to, methylamino, ethylamino,dimethylamino, diethylamino, and the like.

The term “alkyloxy” or “alkoxy”, as used herein, refers to any alkylmoiety attached through an oxygen bridge (e.g., —O-alkyl) such as, butnot limited to, methoxy, ethoxy, and the like.

The term “alkylthio”, as used herein, refers to any alkyl moietyattached through a sulfur bridge (e.g., —S-alkyl) such as, but notlimited to, methylthio, ethylthio, and the like.

The term “alkenyl” refers to an unbranched or branched hydrocarbon chainhaving one or more double bonds therein. The double bond of an alkenylgroup can be unconjugated or conjugated to another unsaturated group.Suitable alkenyl groups include, but are not limited to vinyl, allyl,butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl,2-ethylhexenyl, 2-propyl-2-butenyl,4-(2-methyl-3-butene)-pentenyl. Analkenyl group can be unsubstituted or substituted with one or twosuitable substituents.

The term “alkynyl” refers to unbranched or branched hydrocarbon chainhaving one or more triple bonds therein. The triple bond of an alkynylgroup can be unconjugated or conjugated to another unsaturated group.Suitable alkynyl groups include, but are not limited to ethynyl,propynyl, butynyl, pentynyl, hexynyl, methylpropynyl,4-methyl-1-butynyl, 4-propyl-2-pentynyl-, and 4-butyl-2-hexynyl. Analkynyl group can be unsubstituted or substituted with one or twosuitable substituents.

Compounds and Methods

The present invention relates to selective azophenol ERG inhibitorcompounds and to their use for treating or preventing a disease relatedto over-expression of an ETS Related Gene (ERG), a wild type ERG proteinor an altered ERG protein in a subject. More specifically, the azophenolERG inhibitors of the invention do not attenuate or inhibit androgenreceptor (AR) signaling in ERG negative AR positive CaP cell linestested and thus exhibit fewer toxic side effects when compared toconventional agents that inhibit AR signaling as the underlyingmechanism for treating prostate cancer. Additionally, the azophenols ofthe invention inhibit ERG protein, and cell growth in the ERG positivetumor cell lines that do not express AR.

Approved strategies for the treatment of prostate cancer routinelyentail therapeutic agents that attenuate or inhibit the activity of ARin prostate cancer cells. Because the expression of ERG in VCaP prostatecancer cells is regulated by AR, representative azophenol compounds ofthe invention were evaluated to determine if the observed ERG inhibitoryactivity was a result of AR inhibition. The azophenol compounds wereobserved to inhibit the expression of AR and prostate specific antigen(PSA) in VCaP prostate cancer cells.

To investigate whether the azophenol compounds of the invention areselective inhibitors of ERG protein expression, representative azophenolcompounds were further tested for its ability to inhibit AR and PSAactivity in the following AR positive/ERG negative prostate cancer celllines: LNCaP (mutant AR positive and ERG negative), as well as in LAPC4cells that are AR (wild type) positive and ERG negative.

Inhibition of AR by a representative azophenol compound of the inventionis specific to VCaP cells. No AR inhibition was observed in other ARpositive/ERG negative cell lines used in this screen.

In an exemplary embodiment, a method for treating or preventing adisease related to overexpression of wild type ERG protein or an alteredERG protein product of the E-Related Gene (ERG) in a subject byadministering to the subject a therapeutically-effective amount of anazophenol compound of the invention that selectively inhibits ERGexpression. While the exact mechanism by which ERG expression is loweredor inhibited is unknown, the azophenol compounds may, for example,influence ERG mRNA gene transcription, ERG mRNA translation, prevent ERGprotein from attaining its functionally active tertiary structure orinhibit the growth of ERG positive tumors by altering the regulation ofa gene that is essential for cell growth.

The azophenol compounds of the invention appear to selectively inhibitERG expression in cancer cells without inhibiting the expression of ERGin normal endothelial cells. A representative azophenol compound of theinvention inhibits expression of ERG in a dose dependent manner in ERGpositive cancer cell lines. No measurable inhibition of ERG proteinexpression was observed in normal HUVEC cells, with basal expression ofERG, however.

ERG overexpression in cancer cells is believed to play a role in thedevelopment of oncogene addiction, a condition in which some ERGpositive cancer cells depend on the activity of the ERG protein fortheir growth and survival. Inhibition or attenuation of ERG proteinexpression in ERG positive cancer cells, therefore, may arrest thegrowth and survival of cancer cells. As illustrated by the results of acell growth inhibition study, inhibition of ERG expression preventsgrowth of ERG positive cancer cells. No cell growth inhibition effectswere observed, however, for ERG negative prostate cancer cell lines, anERG negative immortalized benign prostate cell line (BPH1), and for ERGpositive normal cells. These results support the use of the azophenolcompounds of the invention as candidate therapeutic agents for theselective treatment of ERG positive cancers, such as, for example,prostate cancer. In an exemplary embodiment, methods for treating ERGpositive cancers using the azophenol compounds of the inventiontherefore provide an unexpected approach for treating metastatic hormonerefractory prostate cancer that is unresponsive to treatment with agentsthat attenuate or inhibit AR activity and/or ablate hormonal activity.

The azophenol inhibitors of ERG overexpression according to theinvention may also be used in combination with one or more othertherapeutic agents capable of treating cancers. In an exemplaryembodiment, the azophenol compounds are used in combination withconventional inhibitors of AR activity for the treatment of prostatecancer, particularly for subject diagnosed with prostate cancer, inwhich the AR activity is amplified or super activated.

The azophenol compounds of the invention were further evaluated as acandidate therapeutic agent for treating a patient having an ERGpositive cancer. Separate cultures of ERG positive VCaP prostate cancercells and ERG negative LNCaP cells were used to test for selectiveinhibition of ERG expression and the cell growth inhibitory activity ofseveral representative azophenol compounds. The azophenol compounds ofthe invention are selective inhibitors of ERG expression and growth ofERG positive cancer cells.

The above observations and the role of ERG in cancer cell growth supportthe use of ERG specific inhibitors, such the azophenol compounds of theinvention, as viable therapeutic agents for treating cancers such asprostate cancer, colorectal cancer, Ewing sarcoma, a vascular tumor andleukemia. In an exemplary embodiment, the subject receiving treatmentfor cancer according to a method of the invention is a mammal. Forinstance, the methods and uses described herein are suitable for thetreatment of cancers in humans. Alternatively, the methods and uses ofthe invention may be suitable in a veterinary context, wherein thesubject includes, but is not limited to, a dog, cat, horse or cow.

In select embodiments of the invention, the azophenol ERG inhibitors areco-administered with at least one anti-cancer therapeutic agent. As usedherein, “co-administer” indicates that each of the at least twocomponents is administered during a time frame wherein the respectiveperiods of biological activity or effects overlap. Thus, the term“co-administer” is intended to encompass sequential as well ascoextensive administration of the individual therapeutic components.Accordingly, “administering” the combination of components according tosome of the methods of the present invention includes sequential as wellas coextensive administration of the individual components of thepresent invention. Likewise, the phrase “combination of compounds” or“combination of components” and the like indicate that the individualcomponents are coadministered, and these phrases do not necessarily meanthat the compounds must be administered contemporaneously orcoextensively. In addition, the routes of administration of theindividual components need not be the same. In an exemplary embodiment,the azophenol compounds are administered in the same composition.

In an exemplary embodiment, at least one azophenol ERG inhibitor of theinvention is co-administered with a prostate cancer therapy. In a morespecific embodiment, the azophenol ERG inhibitors are co-administeredwith one or more of lutenizing hormone-releasing hormone (LHRH) analogssuch as, but not limited to, leuprolide (Lupron®, Eligard®), goserelin(Zoladex®), triptorelin (Trelstar®), degarelix (Firmagon®), Abiraterone(Zytiga®) and histrelin (Vantas®). In other specific embodiments, theazophenol ERG inhibitors are co-administered with one or more ofanti-androgen receptors such as, but not limited to, flutamide(Eulexin®), bicalutamide (Casodex®), Enzalutamide (Xtandi®) andnilutamide (Nilandron®). In other specific embodiments, the azophenolERG inhibitors are co-administered with one or more chemotherapeuticssuch as, but not limited to, Docetaxel (Taxotere®), Cabazitaxel(Jevtana®), Mitoxantrone (Novantrone®), Estramustine (Emcyt®),Doxorubicin (Adriamycin®), Etoposide (VP-16), Vinblastine (Velban®),Paclitaxel (Taxol®), Carboplatin (Paraplatin®), Vinorelbine (Navelbine®)Abiraterone (Zytiga), ARN-509 (J@J), and Galeterone (Tokai).

In an exemplary embodiment, the azophenol ERG inhibitors areadministered as a first line therapy. In other embodiments, theazophenol ERG inhibitors are administered as a second line therapy or athird line therapy. In still other embodiments, the azophenol ERGinhibitors are administered subsequent to a third line therapy. As usedherein, a first line therapy is the therapeutic regimen that is firstprescribed or followed upon diagnosis of a condition that warrants theuse of an ERG inhibitor, such as but not limited to prostate cancer. Asecond line therapy is the therapeutic regimen that is prescribed orfollowed upon diagnosis of a recurrence or metastasis of condition thatwarrants the use of an ERG inhibitor, such as but not limited toprostate cancer. Likewise, a third line therapy is the therapeuticregimen that is prescribed or followed upon diagnosis of a secondrecurrence or metastasis of condition that warrants the use of an ERGinhibitor, such as but not limited to prostate cancer. A therapy, forthe purposes of determining which “line” of therapy as used herein, neednot be a drug therapy. For example, a first line therapy, as usedherein, may be surgical removal, or radiation therapy. Any therapydesigned to remove, reduce or ablate the tumor or condition can beconsidered a “line” of therapy.

In other embodiments, the azophenol ERG inhibitors of the invention canbe administered as a “maintenance” therapeutic. As used herein, amaintenance therapeutic is a therapeutic regimen that is prescribed orfollowed while the subject is free of any detectable condition requiringtreatment, for example, after a tumor is surgically removed from thesubject. In these embodiments, the ERG inhibitors can be taken, forexample, after surgical resection, for a specified period of time suchas, but not limited to, at least about six months, such as one year, twoyears, three years, four years or five years, after the removal ordisappearance of the tumor or cancer.

Pharmaceutical Formulations, Routes of Administration and Dosing Regimen

Despite evidence generally associating ERG expression with cancer cellgrowth, the conventional art does not appear to consider a smallmolecule compound that selectively inhibits expression of ERG in cancercells or the use of such selective ERG inhibitors as anti-neoplasticagents. The present invention provides azophenol compounds and theirpharmaceutical compositions that are useful in treating a subjectsuffering from an ERG positive cancer, as more generally set forthabove.

The azophenol compound or composition of the invention can be formulatedas described herein and is suitable for administration in atherapeutically effective amount to the subject in any number of ways. Atherapeutically effective amount of an azophenol compound as describedherein depends upon the amounts and types of excipients used, theamounts and specific types of active ingredients in a dosage form, andthe route by which the compound is to be administered to patients.However, typical dosage forms of the invention comprise a compound or apharmaceutically acceptable salt of the compound.

Typical dosage levels for the azophenol compounds generally range fromabout 0.001 to about 100 mg per kg of the subject's body weight per daywhich can be administered in single or multiple doses. An exemplarydosage is about 0.01 to about 25 mg/kg per day or about 0.05 to about 10mg/kg per day. In other exemplary embodiments, the dosage level rangesfrom about 0.01 to about 25 mg/kg per day, such as about 0.05 to about10 mg/kg per day, or about 0.1 to about 5 mg/kg per day.

A dose can typically range from about 0.1 mg to about 2000 mg per dayand can be given as a single once-a-day dose or, alternatively, asdivided doses throughout the day, optionally taken with food. In aparticular embodiment, the daily dose is administered twice daily inequally divided doses. A daily dose range can range from about 5 mg toabout 500 mg per day such as, for example, between about 10 mg and about300 mg per day. In managing the patient, the therapy can be initiated ata lower dose, such as from about 1 mg to about 25 mg, and increased ifnecessary up to from about 200 mg to about 2000 mg per day as either asingle dose or divided doses, depending on the subject's globalresponse.

The azophenol ERG inhibitor compounds according to the invention may bedelivered by oral, parenteral (e.g., intramuscular, intraperitoneal,intravenous, ICV, intracisternal injection or infusion, subcutaneousinjection or implant), inhalation, nasal, vaginal, rectal, sublingual,or topical (e.g., transdermal, local) routes of administration. Theinhibitors can be formulated alone or together, in suitable dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles that are appropriate foreach route of administration.

For example, suitable oral compositions in accordance with the inventioninclude, without limitation, tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, syrups or elixirs. Inventive compositions suitable for oraluse may be prepared according to any method known to the art for themanufacture of pharmaceutical compositions. For example, liquidformulations of the azophenol compounds can contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations of the azophenol ERGinhibitor.

For tablet compositions, typical non-toxic pharmaceutically acceptableexcipients include, without limitation, inert diluents such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents such as, for example,corn starch, or alginic acid; binding agents such as, for example,starch, gelatin or lubricating agents such as, for example, magnesiumstearate, stearic acid or talc. The tablets may be uncoated or,alternatively, they may be coated by known coating techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyto provide a sustained therapeutic action over a desired time period.For example, a time delay material such as glyceryl monostearate orglyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent suchas, for example, calcium carbonate, calcium phosphate or kaolin, or assoft gelatin capsules wherein the active ingredient is mixed with wateror an oil medium such as, for example peanut oil, liquid paraffin orolive oil.

For aqueous suspensions the azophenol compound is admixed withexcipients suitable for maintaining a stable suspension. Examples ofsuch excipients include, without limitation, sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

Oral suspensions can also contain dispersing or wetting agents, such asnaturally-occurring phosphatide such as, for example, lecithin, orcondensation products of an alkylene oxide with fatty acids such as, forexample, polyoxyethylene stearate, or condensation products of ethyleneoxide with long chain aliphatic alcohols such as, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such as, forexample, polyoxyethylene sorbitol monooleate, or condensation productsof ethylene oxide with partial esters derived from fatty acids andhexitol anhydrides such as, for example, polyethylene sorbitanmonooleate. The aqueous suspensions may also contain one or morepreservatives such as, for example, ethyl or n-propyl p-hydroxybenzoate,one or more coloring agents, one or more flavoring agents, and one ormore sweetening agents such as sucrose or saccharin.

Sweetening agents such as those set forth above, and flavoring agentsmay be added to provide palatable oral preparations. These compositionsmay be preserved by the addition of an anti-oxidant such as ascorbicacid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water can provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as, for example, sweetening, flavoring andcoloring agents, may also be present.

Syrups and elixirs may be formulated with sweetening agents such as, forexample, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, and flavoringand coloring agents.

Compositions for parenteral administrations are formulated in a sterilemedium suitable for intravenous, intramuscular or intrathecal delivery.A sterile injectable preparation of the azophenol compounds may be inthe form of a sterile injectable solution or sterile injectablesuspension. Non-toxic, parentally acceptable diluents or solvents suchas, for example, 1,3-butanediol can be used to formulate the parenteralcompositions. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile oils also can be employed as a solvent ora suspending medium. For this purpose, any bland fixed oil may beemployed, including synthetic monoglycerides or diglycerides. Inaddition, fatty acids such as oleic acid can be used in the preparationof injectables.

Depending on the vehicle used and the concentration of the drug in theformulation, the parenteral formulation can contain other adjuvants suchas local anesthetics, preservatives and buffering agents.

EXAMPLES

Cell Lines

Tumor cell lines VCaP, COLO320, KG-1, MOLT4, LNCaP, and MDA Pca2b wereobtained from the American Tissue Culture Collection (ATCC; Manassas,Va.). The cells were grown in ATCC-recommended cell culture media undercell growth promoting conditions as recommended by the supplier. Normalcells, such as HUVEC-primary cultures of human umbilical veinendothelial cells and the RWPE1 cell line established from normal adultprostate epithelial cells immortalized with human papilloma virus 18were also obtained from ATCC. The BPH1 cell line derived from primaryepithelial cell cultures immortalized with SV40 large T-antigen, were agift from Dr. Simon Hayward (Vanderbilt University Medical Center).LAPC4, a metastatic prostate cancer cell line was a gift from Dr.Charles Sawyer (then at UCLA).

Reagents

ERG monoclonal antibody (CPDRERG-MAb; 9FY, licensed to Biocare Medical,CA) was developed and characterized at the Center for Prostate DiseaseResearch. Antibodies for the androgen receptor (AR; sc-816),glyceraldehyde phosphate dehydrogenase (GAPDH; sc-25778), and α-Tubulin(sc-5286) were purchased from Santa Cruz Biotechnology (Santa Cruz,Calif.). Antibody for prostate specific antigen (PSA; A0562012) wasobtained from DakoCytomation (Carpinteria, Calif.). Antibodies forapoptosis (9915S) and cell cycle regulator (9932) sampler kits werepurchased from Cell Signaling (Danvers, Mass.). Sheep anti-mouse IgG-HRP(NXA931) and donkey-anti rabbit IgG-HRP (NXA934V) were obtained from GEHealth Care, Buckinghamshire, UK. A number of the azophenol compoundswere obtained from the Stanford University School of Medicine. Many ofthe azophenol compounds disclosed herein are known in the prior art,typically for their use as dyes. The published methods for theirsyntheses represent routes by which the compounds of the invention wereprepared.

General Protocol for Screening Inhibitors of the ERG OncoproteinExpression

The TMPRSS2-ERG fusion positive prostate cancer cell line, VCaP (ATCC),was used to identify the test compound inhibitors of ERG expression.Cells were grown in medium using conditions prescribed by the vendor.VCaP cells in logarithmic growth were seeded in a tissue culture dish ata cell density of 2×10⁶ cells per plate. Following 48 hours incubationat 37° C., cells were exposed for a period of 48 hours with 0, 0.05,0.25 and 0.5 μM concentrations of each test azophenol compound. Theinhibition of ERG expression was evaluated by an In-cell Western blotassay (LI-COR Biosciences, Lincoln, Nebr.) using the ERG specific CPDRERG-MAb as further described below.

Selection of ERG siRNA as a Positive Control

Small interfering RNA (siRNA) oligo duplexes (5′ CGA CAU CCU UCU CUC ACAUAU 3′: si-1; and 5′ UGA UGU UGA UAA AGC CUU A 3′: si-2) against humanERG gene (Gene ID: 2078; Accession: NM 004449), were purchased fromDharmacon (Lafayette, Colo.) and were evaluated as positive controls foruse in the ERG expression inhibition screens. Two siRNAs were chosen toprimarily rule out off target or non-specific effects. Since both siRNAsshowed identical results, si-1 was used in the ERG expression inhibitorystudies described below. A non-targeting (NT) siRNA duplex was used asnegative control (D-001206-13-20; Dharmacon, Lafayette, Colo.). Cellswere cultured in their respective growth medium for 48 hours prior totransfection using a 50 nM concentration of the NT siRNA or ERG siRNA.Lipofectamine 2000® (Invitrogen, Carlsbad, Calif.) was used fortransfection.

General Protocol for Evaluating the Inhibitory Effects of Test Compoundsby Western Blot Analysis

Inhibition of ERG protein expression by the test compounds weredetermined by Western blot analysis. The ERG specific monoclonalantibody CPDR ERG-MAb was used as the primary antibody. In brief,Western blot was performed by running a fixed amount of total proteinextracted from cell lysates of the treated cells using (4-12% Bis-Tris)gel by electrophoresis, followed by transfer to membrane and incubationwith primary antibody and continued with HRP-conjugated secondaryantibody. Cultured cells were treated at specific dosages with each ofthe tested ERG inhibitors. Following incubation of the treated cells foran indicated time period, cells were lysed using Mammalian ProteinExtraction Reagent (M-PER; Pierce, Rockford, Ill.) containing a proteaseinhibitor cocktail and phosphatase inhibitor cocktails I & III (Sigma,St Louis, Mo.). Cell lysates containing 50 μg of total protein wereelectrophoresed through 4-12% Bis-Tris Gel (Invitrogen, Carlsbad,Calif.) and the cellular proteins were transferred to PVDF membrane(Invitrogen, Carlsbad, Calif.). Membranes were incubated at 4° C. for 12hours with primary antibodies for AR, PSA, GAPDH, α-Tubulin, apoptosismarkers and cell cycle regulators. Following exposure to primaryantibodies, the membranes were washed with buffer (3×, 5 minutes each atroom temperature) followed by incubation with relevant secondaryantibodies for 1 hour at 24° C. Finally, the membranes were washed withbuffer and developed using the ECL Western blot detection reagent (GEHealth Care, Buckinghamshire, UK). The ERG protein expression of thetest azophenol compounds were normalized with GAPDH.

Selective Inhibition of ERG Expression

In brief, ERG positive VCaP cells in logarithmic growth phase wereplated in 10 cm tissue culture dish at a cell density of 2×10⁶ cells perdish. The plated cells were treated with 0, 0.2, 0.4, 0.6, 0.8 and 1 μMconcentrations of each azophenol compound for a period of 48 hours.Cells from each dish were then processed for Western blot analysis andalterations in the expression of ERG protein were monitored. Both ERGand GAPDH protein band were quantified from each concentration usingImage J (NIH) and ERG band density was normalized with correspondingGAPDH protein control. Relative density of ERG in each concentration wascalculated and with Graphpad Prism 6 software, the IC50 of each compoundwas calculated.

General Protocol for Cell Growth and Tumor Growth Inhibition Studies

The appropriate ERG positive cancer cells, control ERG negative cells orERG positive normal cells were grown as adherent monolayers orsuspensions in tissue culture dishes using the appropriate growth mediumas suggested by the vendor. Approximately 48 hours following plating ofcells, the appropriate test compound is added to each well of the tissueculture dish at a predetermined concentration. The medium wasreplenished every 24 hours with fresh growth medium containing the sameconcentration of the same test compound for indicated period of the cellgrowth inhibition assay. Percent cell growth inhibition was calculatedusing a hemocytometer for estimating cell density in each of the testwells of the tissue culture dish and trypan blue dye exclusionmicroscopy and photography to estimate the fraction of viable cells ineach test well.

To investigate whether the azophenol compounds selectively arrested thegrowth of ERG positive cancer cells (VCaP), VCaP cells were cultured toachieve cells in logarithmic growth and these cells were then plated in6 well tissue culture dishes in duplicates at a cell density of 0.2×10⁶cells per well. The plated cells were exposed to 0, 0.2, 0.4, 0.6, 0.8and 1 μM concentrations of each compound for a period of 8 days. At theend of time period, cells were recovered from the test plate, washed,trypsinized and the cell density of viable cells were determined withtrypan blue dye staining method and automated cell counter Bio-Rad TC10.The IC50 of cell growth inhibition of each compound was determined bytrypsinizing the cells at the end of the experiment and counting thecells by using the automated cell counter (Bio-Rad TC10 automated cellcounter). The average cell numbers of each concentration were used tocalculate the IC50 of each compound with the GraphPad Prism 6 software.

Male athymic nude mice 6-8 weeks old and weighing 27 to 30 g werepurchased from Charles River Laboratories. ERG harboring prostate cancercells (VCaP) were trypsinized and washed twice with ice-cold PBS, andresuspended in ice-cold 50% matrigel in serum-free DMEM medium. A totalof 4×10⁶ cells/0.1 mL/mouse were subcutaneously injected into lowerright dorsal flank of the mice. Prior to injection, mice wereanesthetized with inhalation anesthesia (isoflurane). Tumor growth wasmonitored weekly after injection. Three weeks later when tumors werepalpable mice were randomly separated into 2 experimental groups and onecontrol group of 7 mice in each group. In the treatment groups, micewere injected intraperitoneally (LP) with 100 mg/kg of the test compoundor 150 mg/kg of the test compound while the control group were injectedwith vehicle (1:1[v/v], DMSO/PEG300) only. Growth in tumor volume wasrecorded weekly by digital caliper measurements and tumor volumescalculated using the ½ (L×W²) formula, where L=length of tumor andW=width. Tumor volumes were compared between treated and control groupswith repeated measurements and statistical significance of the resultsbetween the groups computed using students t-test and p valuescalculated.

Table 1 below shows the results of testing of azophenol compounds.

ERG protein Cell growth inhibition inhibition Compound Cell line Tissueorigin ERG gene status (IC50) (IC50) 3 VCaP Metastatic TMPRSS2-ERGfusion; ERG 1.352  4.070  prostate cancer protein (N-33 aa-del) 4 VCaPMetastatic TMPRSS2-ERG fusion; ERG 1.855  1.122  prostate cancer protein(N-33 aa-del) 8 VCaP Metastatic TMPRSS2-ERG fusion; ERG N/D N/D prostatecancer protein (N-33 aa-del) 9 VCaP Metastatic TMPRSS2-ERG fusion; ERGN/D N/D prostate cancer protein (N-33 aa-del) 10 VCaP MetastaticTMPRSS2-ERG fusion; ERG N/D N/D prostate cancer protein (N-33 aa-del) 5VCaP Metastatic TMPRSS2-ERG fusion; ERG 0.5422 0.5572 prostate cancerprotein (N-33 aa-del) 6 VCaP Metastatic TMPRSS2-ERG fusion; ERG 0.27270.0739 prostate cancer protein (N-33 aa-del) 7 VCaP MetastaticTMPRSS2-ERG fusion; ERG 0.5587 0.6260 prostate cancer protein (N-33aa-del) 11 VCaP Metastatic TMPRSS2-ERG fusion; ERG N/D N/D prostatecancer protein (N-33 aa-del) 12 VCaP Metastatic TMPRSS2-ERG fusion; ERGN/D N/D prostate cancer protein (N-33 aa-del) 13 VCaP MetastaticTMPRSS2-ERG fusion; ERG N/D N/D prostate cancer protein (N-33 aa-del)N/D: Assay performed, inhibition not detected at the tested levels.

All publications cited herein are incorporated by reference in theirentireties.

REFERENCES

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We claim:
 1. A method for treating a disease associated withoverexpression of wild type ERG protein, an altered ERG protein, ERGgene transcription or ERG mRNA translation selected from the groupconsisting of prostate cancer and colon cancer in a subject sufferingtherefrom, comprising administering to the subject a therapeuticallyeffective amount of a compound selected from the group consisting ofCompounds 1-7:

or a pharmaceutically acceptable salt thereof.
 2. The method accordingto claim 1, wherein the disease is prostate cancer.
 3. The methodaccording to claim 1, wherein the disease is colon cancer.
 4. The methodaccording to claim 1, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 5. Themethod according to claim 1, wherein the compound is Compound
 1. 6. Themethod according to claim 1, wherein the compound is Compound
 2. 7. Themethod according to claim 1, wherein the compound is Compound
 3. 8. Themethod according to claim 1, wherein the compound is Compound
 4. 9. Themethod according to claim 1, wherein the compound is Compound
 5. 10. Themethod according to claim 1, wherein the compound is Compound
 6. 11. Themethod according to claim 1, wherein the compound is Compound
 7. 12. Themethod according to claim 5, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 13. Themethod according to claim 6, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 14. Themethod according to claim 7, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 15. Themethod according to claim 8, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 16. Themethod according to claim 9, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 17. Themethod according to claim 10, wherein the compound is present in apharmaceutical composition further comprising an excipient.
 18. Themethod according to claim 11, wherein the compound is present in apharmaceutical composition further comprising an excipient.